Selected-sequence biopolymers, such as polypeptides and polynucleotides, are routinely synthesized by solid-phase methods in which a series of selected polymer subunits are added sequentially to a growing polymer chain carried on a solid support. In a typical polypeptide synthesis, the polymer is synthesized stepwise from an immobilized C-terminal residue. At each step, a new N-protected amino acid is added in solution to the solid support, and reacted through its free carboxyl group with the free .alpha.-amine group of the amino acid (or peptide) immobilized on the support, to couple the new amino acid to the growing peptide on the support. The support is then treated to remove the N-protecting group of the last added amino acid, and the procedure is repeated in a stepwise fashion until the final polypeptide is complete.
A polynucleotide, e.g., DNA strand, is similarly synthesized by solid-phase methods, by stepwise addition of a selected 5'-protected nucleotide to a resin containing an immobilized 3'-end nucleoside. Backbone coupling is between the free 5' OH group of the immobilized nucleoside, and the activated 3'-end of the free nucleotide. After the coupling reaction, the support is treated to remove the 5'-end protecting group, and the reaction steps are repeated stepwise until the desired-sequence polynucleotide is complete.
Solid-phase methods for polypeptide and polynucleotide synthesis can be carried out conveniently by automated synthesizers which are designed for successive addition of selected subunits, coupling agents, and deprotecting agents to a vessel containing the solid-phase material. That is, each subunit addition step involves (a) adding a deprotection solution to the solid-phase vessel, to deprotect the last-added residue on the immobilized support, and (b) adding the next subunit, either in activated form or in the presence of an activator, to the solid-phase vessel, to couple the subunit to the growing polymer chain on the solid support.
In a typical operation of an automated synthesizer, the machine is first loaded with vials containing each of the subunits which are to be added during operation, and with the deprotection and wash solutions used during operation. The vials containing the individual subunits may be prepackaged in liquid form, allowing the subunit solution to be transferred readily from the vial to the solid-phase vessel. Such vials, of course, must be stored in a manner which prevents leakage or breakdown of the subunit or activation components.
Alternatively, the vials may be packaged in dried form. The dried material is either manually dissolved prior to loading into the machine, or more commonly, is dissolved during machine operation, by addition of a selected volume of solvent to each vial. One limitation of the dried material is that, for many amino acids, a low bulk density of the material makes the material difficult to measure and package. In addition, some dried amino acids dissolve very slowly and may require up to thirty minutes of contact time with an added solvent before the subunit is fully dissolved. For these reasons, a composition having handling and dissolution properties which are superior to those of the existing available reagents would be useful.
Automated apparatus has been built for synthesis of polypeptides and polynucleotides as described above, but there are many problems that have never been adequately overcome. Among them are a need for ease of use, relatively low instrument cost, and low cycle costs.
The need for ease-of-use relates to a developing market in which users of synthesizers are not experienced peptide and polynucleotide chemists, but increasingly are immunologists, neurobiologists, and molecular biologists, among other disciplines. Lacking the particular experience of peptide chemists, these new users need relatively more automatic and trouble-free operation than has been previously provided.
There is also a perceived need for a relatively small scale instrument, capable of ordering the synthesis of chains of thirty subunits or so, adequate for most requirements of the new class of users. At the same time, since the use of such an instrument may not be as extensive as it might be for an instrument in a research lab devoted to full-time preparation of macromolecules by peptide and polynucleotide chemists, there is a greater need for low initial cost, and certainly a greater need for low cycle costs. The cycle costs are the costs for subunit materials, reagents, and the like, used in the synthesis operations.
Another need not yet adequately met, that relates to the use of such an instrument by researchers not particularly experienced in peptide and polynucleotide synthesis, is the need for monitoring the reactions involved. It is known, for example, that coupling reactions are sometimes unexpectedly slow, and it has been observed that slow coupling reactions in peptide synthesis are often preceded by slow deprotection steps.
What is needed then, in addition to a composition for subunit materials having handling and dissolution properties which are superior to those of existing available reagents, is a relatively low-cost, very reliable instrument capable of synthesizing molecules of moderate length (30 subunits or so), with monitoring capability coupled with automatic adjustment of timing for coupling steps. In addition the instrument needs to be very reliable and easy to use.